Homologous recombination using targeting vectors that are specifically designed to add, delete, or replace a particular nucleic acid sequence at a genomic locus is a popular approach to achieving a desired genomic modification in non-human animals. A nuclease that is specifically engineered to introduce a singe or a double-strand break at or near a target genomic locus can be used together with a targeting vector to enhance efficiency of homologous recombination at the target genomic locus.
Although the art of genome modification through homologous recombination has advanced considerably over the last two decades, difficulties still remain with achieving an acceptable targeting frequency using very large targeting vectors, LTVECs, in many circumstances, for example, when a large portion of a rodent genome is replaced with a large human genomic fragment, or difficulties targeting certain cell types, e.g., fibroblasts or other somatic cells. There is a need in the art for further and improved methods for modifying large genomic loci of a eukaryotic genome using LTVECs.